OBJECTIVE To determine the location of NRA-projecting neurons in the periaqueductal gray (PAG), and to determine whether the projection from the PAG to the nucleus retroambiguus is monosynaptic. The periaqueductal gray (PAG) is known to be essential for vocalization and reproductive behavior. The PAG controls components of these behaviors via projections to the nucleus retroambiguus (NRA), a group of premotor neurons in the lower brainstem. The NRA and its lumbosacral projections have recently been identified in the rhesus monkey, but there is no information about the PAG-NRA connection in primates. In the present light and electron microscopical tracing study, wheatgerm agglutinin horseradish peroxidase (WGA-HRP) was injected into the NRA in six monkeys to locate midbrain neurons projecting to the NRA. In three monkeys, the distribution pattern of PAG axons in the brainstem was determined by making WGA-HRP injections into the PAG and adjacent tegmentum. In one of these three monkeys, biotinylated dextran amine and choleratoxin subunit b were injected into the lumbosacral cord to identify NRA neurons. The results showed that a compact group of neurons in the central part of th e lateral PAG at the intercollicular level send a dense projection to the NRA. There existed monosynaptic contacts between the PAG and NRA neurons, including NRA neurons that project to the lumbosacral cord. The synaptic contacts were asymmetrical and contained spherical and dense core vesicles. It can be concluded that there exists a strong and direct PAG-NRA pathway in the rhesus monkey. Since NRA neurons projecting to the lower lumbar cord are included, the PAG-NRA projection is likely to be involved not only in vocalization, but also in other behaviors such as receptive posture. FUTURE DIRECTIONS We will examine the effects of estrogen on this pathway. KEY WORDS nucleus retroambiguus, periaqueductal gray, neuronal tracing, electron microscopy, vocalization, sexual behavior FUNDING Supplemental funding to RR00167